Improving sequence-specific antimicrobials by blocking dna repair

Patent metadata

Field	Value
Publication number	US-2018200342-A1
Application number	US-201615744039-A
Country	US
Kind code	A1
Filing date	Jul 13, 2016
Priority date	Jul 13, 2015
Publication date	Jul 19, 2018
Grant date	—

Abstract

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

First claim

1 - 27 . (canceled)

28 . A method for killing a bacterium comprising contacting the bacterium with at least one recombinant phagemid(s) or plasmid(s);
wherein the recombinant phagemid(s) or plasmid(s) encodes an endonuclease that creates a double-stranded break (DSB) in the chromosomal or extrachromosomal DNA of the bacterium, and an exogenous protein that inhibits DSB repair.

29 . The method of claim 28 , wherein the exogenous protein is encoded by the same vector as the endonuclease or by a separate vector.

30 . The method of claim 28 , wherein the protein is synthetized before contacting with the bacterium.

31 . The method of claim 28 , wherein the endonuclease is selected from a meganuclease or an artificial endonuclease.

32 . The method of claim 28 , wherein the endonuclease specifically cleaves the chromosomal or extrachromosomal DNA of the bacterium at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different sites.

33 . The method of claim 28 , wherein the at least one recombinant phagemid(s) or plasmid(s) encodes a Cas9 nuclease, a guide RNA, and an exogenous protein that inhibits DNA repair selected from the group consisting of a Mu phage Gam protein, a lambda phage Gam protein, and a phage T7 gp5.9 protein.

34 . The method of claim 28 , wherein the at least one recombinant phagemid(s) is selected from the group consisting of M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2, 13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37, MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage, Pseudomonas phage ϕ6, ϕ29 phage and 186 phage.

35 . The method of claim 28 , wherein the bacterium comprises a recBCD homologous repair pathway or addAB system.

36 . The method of claim 28 , wherein the bacterium is selected from the group consisting of Enterobacter, Streptococci, Staphylococci, Enterococci, Salmonella, Pseudomonas , and Mycobacterium.

37 . The method of claim 28 wherein the recombinant phagemid(s) or plasmid(s) encode(s) an endonuclease that creates a double-stranded break (DSB) in an antibiotic resistance gene encoded by the bacterium

38 . λ phagemid or plasmid vector encoding an endonuclease and an exogenous protein inhibiting DSB repair.

39 . The phagemid or plasmid vector of claim 38 wherein the recombinant phagemid(s) is selected from the group consisting of M13, lambda, p22, T7, Mu, T4 phage, PBSX, P1Puna-like, P2, 13, Bcep 1, Bcep 43, Bcep 78, T5 phage, phi, C2, L5, HK97, N15, T3 phage, P37, MS2, Qβ, or Phi X 174, T2 phage, T12 phage, R17 phage, M13 phage, G4 phage, Enterobacteria phage P2, P4 phage, N4 phage, Pseudomonas phage ϕ6, ϕ29 phage and 186 phage.

40 . The phagemid or plasmid vector of claim 38 , wherein the phagemid vector is a P1 bacteriophage.

41 . The phagemid or plasmid vector of claim 38 , wherein the phagemid vector is a λ bacteriophage.

42 . A pharmaceutical composition comprising a phagemid or plasmid vector encoding an endonuclease, and an exogenous protein inhibiting DSB repair or a vector encoding an exogenous protein inhibiting DSB repair, and a pharmaceutically acceptable vehicle.

43 . The pharmaceutical composition of claim 42 further comprising an antibiotic.

44 . The pharmaceutical composition of claim 42 containing a phagemid or plasmid vector encoding an endonuclease and a vector encoding an exogenous protein inhibiting DSB repair.

45 . The pharmaceutical composition of claim 42 , wherein said exogenous protein is encoded by the same vector as the endonuclease.

Assignees

• Pasteur Institut
• Eligo Bioscience

Inventors

• Bikard David
• Cui Lun
• Duportet Xavier
• Fernandez Rodriguez Jesus

Classifications

• A61K38/465 Primary

  acting on ester bonds (3.1), e.g. lipases, ribonucleases · CPC title

• C12N15/1024

  In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• A61P31/04

  Antibacterial agents · CPC title